DE69526577T2 - NEW OPIOID PEPTIDES FOR THE TREATMENT OF PAIN AND USE THEREOF - Google Patents

Abstract

This invention relates to a series of novel opioid peptides for the treatment of pain and pharmaceutically acceptable compositions comprising those peptides. The invention also teaches methods for controlling pain in patients using compositions of the invention. The peptides of this invention have a high degree of selectivity for the mu -opioid receptor. These peptides are highly lipophilic. In spite of their lipophilic character they do not readily cross the blood brain barrier (BBB). The peptides of the present invention are particularly well-suited as analgesic agents acting substantially on peripheral mu -opioid receptors. Because these peptides act peripherally, they substantially avoid producing side effects normally associated with central analgesic action.

Description

Background of the invention

Many endogenous peptides derived from mammals and amphibians bind to specific opioid receptors and elicit an analgesic response similar to classical narcotic opiates. Many different types of opioid receptors have been shown to coexist in higher animals; see, for example, W. Martin et al., J. Pharmacol. Exp. Ther., 197, p. 517 (1975); and J. Lord et al., Nature (London), 257, p. 495 (1977). Three different types of opioid receptors have been identified. The first type, δ, shows a differential affinity for enkephalin-like peptides. The second type, u, shows enhanced selectivity for morphine and other polycyclic alkaloids. The third type, κ, shows equal affinity for both groups of the above ligands and a preferential affinity for dynorphin. In general, μ receptors appear to be more involved in analgesic effects. The δ receptors appear to involve behavioral effects, although the δ and κ receptors can also mediate analgesia.

Each opioid receptor, when coupled with an opiate, elicits a specific biological response that is unique to that type of receptor. If an opiate activates more than one receptor, then the biological response for each receptor is affected, causing side effects. The less specific and selective an opiate is, the greater the likelihood that increased side effects will be elicited by administration of the opiate.

In the prior art, opiates, opioid peptides and analogues thereof have either demonstrated no specificity and selectivity for the type of receptor or receptors to which they bind, or they have demonstrated a limited degree of specificity and selectivity.

The main site of action of analgesic opioids is the central nervous system (CNS). Conventional narcotic analgesics are usually quite hydrophobic, and they are therefore particularly well suited to penetrating lipid membranes, such as the blood-brain barrier. Due to this physical property, analgesics tend to bind to opioid receptors within the central nervous system in the brain. They bind However, it does not necessarily bind to a homogeneous receptor subtype. This binding causes the occurrence of medically undesirable side effects.

Opiates can cause serious and potentially fatal side effects. Side effects such as respiratory distress, habituation, physical dependence and increased withdrawal symptoms are caused by non-specific interactions with receptors of the central nervous system; see K. Budd, in: International Encyclopedia of Pharmacology and Therapeutics; N. E. Williams and H. Wilkinson, eds., Pergammon: (Oxford), 112, p. 51 (1983). It would therefore be expected that opioid analgesics that act primarily through opioid receptors in the peripheral nervous system would not cause similar undesirable side effects as those associated with opioid analgesics that affect the central nervous system. The opioid peptides of the invention affect essentially the peripheral nervous system and thus overcome some of the disadvantages of conventional opiates by substantially avoiding the occurrence of undesirable side effects.

To date, one of the few classes of agents known to produce peripheral analgesic effects are the non-steroidal anti-inflammatory agents, such as aspirin, ibuprofen and ketorolac. These agents do not interact with opioid receptors, but are known to inhibit cyclooxygenase and attenuate prostaglandin synthesis. These weak analgesics have no centrally mediated side effects, but may produce other side effects, such as ulceration of the gastrointestinal tract. An object of the present invention is to provide opioid-like peptide compounds that act peripherally, but substantially avoid the undesirable side effects associated with conventional peripherally acting analgesics.

It has recently been demonstrated in the art that there is significant peripheral analgesic activity of opiate drugs; see A. Barber and R. Gottschlich, Med. Res. Rev., 12, p. 525 (1992) and C. Stein, Anasth. Analg., 76, p. 182 (1993). Quaternary salts of known centrally acting opioid alkaloids have been used as pharmacological probes to distinguish between peripheral and central analgesic responses. The quaternary salts of potent opiates have a permanent positive charge and show limited penetration of the blood-brain barrier; see TW Smith et al., Life Sci., 31, p. 1205 (1982); TW Smith et al., Int. J. Tiss. Reac., 7, p. 61 (1985); BB Lorenzetti and SH Ferreira, Braz. J. Med. Biol. Res., 15, p. 285 (1982); DR Brown and LI Goldberg, Neuropharmacol., 24, p. 181 (1985); G. Bianchi et al., Life Sci., 30, p. 1875 (1982); and J. Russel et al., Eur. J. Pharmacol., 78, p. 255 (1982). Highly polar analogues of enkephalins and dermorphins which retain high antinociceptive activity but show limited penetration into the central nervous system have been prepared; cf. RL Follenfant et al., Br. J. Pharmacol., 93, p. 85 (1988); GW Hardy et al., J. Med. Chem., 32, p. 1108 (1989). On the other hand, it has been assumed in the prior art that lipophilic opioid peptides penetrate the blood-brain barrier more easily. Surprisingly, the opioid peptides of the invention are highly lipophilic, but do not penetrate the blood-brain barrier to any significant extent.

Unlike traditional opiates, opioid peptides are hydrophobic. Their hydrophobic nature tends to increase the rate of their elimination from the mammalian body. The hydrophobic nature enhances the ability of these peptides to cross epithelial barriers. Notwithstanding, administration of opioid peptides to the mammalian body has been shown to affect the central nervous system. Therefore, great efforts have been made to improve the absorption properties of these compounds. Scientists have tried to mitigate the penetration of the peptides into the central nervous system, especially when prolonged exposure of the body to these chemicals could cause undesirable side effects or even be toxic.

It has been suggested that non-polar peptides penetrate the central nervous system more easily than polar peptides by crossing the blood-brain barrier. TAPP (H-Tyr-D-Ala-Phe-Phe-NH2) has been published to exhibit central antinociceptive properties (P. Schiller et al., Proceedings of the 20th European Peptide Symposium, 1988). In contrast, this tetrapeptide TAPP (H-Tyr-D-Ala-Phe-Phe-NH2) has been found by the inventors of the present application to not act centrally. This result was demonstrated by the lack of analgesic effects even at doses of 100 mg/kg in the mouse hot plate test. This test is standard and well known to those skilled in the art. The test detects chemicals that elicit a centrally mediated analgesic response.

Schiller et al. in PNAS, 89, 11871-76 (1992) disclose opioid peptide analogues consisting entirely of aromatic amino acid residues and containing conformationally constrained phenylalanine derivatives in position 2 of the peptide sequence.

The term "specificity" as used in the present application refers to the specific or definitive binding of an opiate or opioid peptide to a specific opioid receptor over another opioid receptor. The specificity of an opioid peptide is indicated by the binding inhibition constant, Ki. The term "selectivity" refers to the ability of an opiate or opioid peptide to discriminate between multiple opioid receptors and to bind only to a specific receptor. The selectivity of an opioid peptide for the u receptor is indicated by the ratio of the binding inhibition constants. For example, the ratio of the binding inhibition constants Kiδ/Kiu is one value that can be used to measure selectivity. This ratio represents the relationship of affinities for binding to u and δ receptors. A higher value for this ratio indicates a stronger preference for binding to the u receptor over the δ receptor by the ligand. A conventional opioid peptide analogue, namely H-Tyr-D-Ala-Gly-Phe(NMe)-Gly-ol (DAGO), is known to be one of the most u-selective opioid peptide analogues. The peptide shows a Kiδ/Kiu value of 1050. Leu-enkephalin, on the other hand, shows a K1δ/Kiu value of 0.2. This fractional value reflects a pronounced affinity for the δ receptor over the u receptor.

A peptide must have certain properties to be pharmacologically useful. First, a peptide should be resistant to proteolytic degradation. Second, a peptide should elicit an enhanced biological response. Third, a peptide must be safe for human consumption. Fourth, a peptide should be suitable for synthesis in sufficiently large quantities for use in clinical trials to consider its toxicity and later for commercialization. In the present case, lower lipid solubility and higher water solubility are also properties that the peptide would advantageously possess to prevent passage through the blood-brain barrier and to allow rapid excretion of any excess peptide and its metabolites administered. Furthermore, it would be advantageous if the peptide elicited selective and specific receptor binding activity to minimize possible side effects.

There is a need for peptides that act on a specific opioid receptor, especially the u-receptor. It would be desirable to find peptides with lower lipid solubility than that of conventional opiates, so that the blood-brain barrier would not be breached. Furthermore, peptides with high polarity would normally be more soluble in aqueous media at physiological pH, enhancing their excretion and the excretion of their metabolites.

Summary of the invention

The present invention provides novel compounds that are selective and specific for substantially one opioid receptor. The present invention provides peptides that exhibit preferential selectivity and specificity for the u-opioid receptor. The invention also provides peptides that interact primarily with opioid receptors on peripheral nerve endings and do not substantially cross the blood-brain barrier. The present invention therefore reduces the severity and number of side effects compared to the side effects associated with conventional opiates and opioid receptor peptides reported to date.

The compounds of the invention are represented by the formula (1):

H-Tyr-R2 -Phe-Phe-NH2 (1)

where:

R&sub2; is D-serine, D-arginine or D-norvaline;

or a pharmaceutically acceptable salt thereof.

The invention also provides pharmaceutically acceptable compositions containing the peptides for use in the treatment of pain.

The invention also provides the use of the peptides for the manufacture of peripheral analgesics for use in the treatment of pain.

The invention further provides the use of a peptide of the formula H-Tyr-D-Ala- Phe-Phe-NH₂ for the manufacture of a peripheral analgesic for use in the treatment of pain.

Description of the invention

The following common abbreviations are used in the description and claims:

Abu - Aminobutyric acid

Aib - Aminoisobutyric acid

Ala - Alanine

Chl - Cyclohomoleucine

Arg - Arginine

Cys (Bzl) - Cysteine (Benzyl)

Cle - Cycloleucine

Dmt - 2',6'-dimethyltyrosyl

Gln - Glutamine

Glu - Glutamic acid

Gly - Glycine

GPI - Guinea pig ileum

His - Histidine

Ile - Isoleucine

Hph - Homophenylalanine

Met - Methionine

Leu - Leucin

MVD - mouse vas deferens

Nle - Norleucin

Nva - Norvalin

Phe - Phenylalanine

Pro - Proline

Phg - Phenylglycin

Ser - Serine

Thr - Threonine

Trp - Tryptophan

Tyr - Tyrosine

Nal - 1'- or 2'-naphthylalanine

PBQ - Phenyl-p-benzoquinone

Tic - Tetrahydroisoquinoline-3-carboxylic acid

TAPP - H-Tyr-D-Ala-Phe-Phe-NH2

TSPP - H-Tyr-D-Ser-Phe-Phe-NH2

The terms "amino acid" and "aromatic amino acid" as used herein include naturally occurring amino acids as well as non-natural amino acids, their derivatives and analogs commonly used by those skilled in the art of chemical synthesis and peptide chemistry. Also included are analogs of TAPP in which the phenylalanine is para-substituted at position 4 with a nitro or azido residue. A list of non-natural and non-proteogenic amino acids can be found in "The Peptide", Vol. 5, 1983, Academic Press, Chapter 6 by D. C. Roberts and F. Vellaccio. Examples of aromatic amino acids include tyrosine, tryptophan, phenylglycine, histidine, naphthylalanine, tetrahydroisoquinoline-3-carboxylic acid and benzylcysteine. Further examples of aromatic amino acids include phenylalanine substituted on the aromatic ring with, for example, CH3, C2H5, F, Cl, Br, NO2, OH, SH, CF3, CN, COOH and CH2COOH or substituted on the β-carbon with a lower alkyl radical, OH, SH or a benzyl group. The aromatic ring can be polysubstituted. Aromatic amino acids can also include aromatic carbocycles of the phenylglycine type, wherein the aromatic ring of the phenylglycine is substituted with CH3, C2H5, F, Cl, Br, NO2, OH, SH, CF3, CN, COOH and CH2COOH.

The term "ED50" as given in Table 1 for the PBQ writhing assays is defined as the dose of drug that induces a 50% reduction in the number of writhes observed compared to the control. The term "ED50" used in the hot plate assays is defined as the dose of drug required to increase the latency of the response two-fold compared to controls and was determined by parallel line probit analysis.

The term "Ki" is the binding inhibition constant. The term "Kiδ/Kiu" is a value used to measure selectivity. This ratio represents the relationship of the affinities of opioid peptides for binding to u and δ receptors.

The term "R configuration" refers to the three-dimensional arrangement of substituents around a chiral element. A general system for designating the absolute configuration is based on a priority system that is well known to those skilled in the art and is briefly described below. Each group associated with a chiral center is assigned a number according to the priority.

The molecule is viewed from the side opposite to the lowest priority. The configuration is called "R" if the eye moves clockwise from the highest priority group to the lowest priority group.

The term "residue" when applied to an amino acid means a group which is derived from the corresponding amino acid by removal of the hydroxyl of the carboxyl group and a hydrogen of the amino group.

Compounds according to the invention are represented by the formula (1):

H-Tyr-R2 -Phe-Phe-NH2 (1)

where:

R&sub2; is D-serine, D-arginine or D-norvaline;

or a pharmaceutically acceptable salt thereof.

The novel compounds according to the invention are given below:

H-Tyr-D-Nva-Phe-Phe-NH2

H-Tyr-D-Ser-Phe-Phe-NH2

H-Tyr-D-Arg-Phe-Phe-NH2

The best mode currently known for carrying out the invention is to use the compound H-Tyr-D-Arg-Phe-Phe-NH₂.

The invention also includes the use of the compound TAPP H-Tyr-D-Ala- Phe-Phe-NH₂ as a peripheral analgesic.

As shown in Table 1, the ED50 value for TAPP (BCH1774) is 1.4. The corresponding values for H-Tyr-D-Nva-Phe-Phe-NH2 (BCH2462), H-Tyr-D-Ser-Phe-Phe-NH2 (BCH2463) and H-Tyr-D-Arg-Phe-Phe-NH2 (BCH2687) are 2.7, 0.5 and 0.5, respectively. The ED50 value for the remaining reference compound is higher than these numbers.

Pharmaceutically acceptable salts of the peptides of the invention can be prepared in a conventional manner by reaction with a suitable acid. Suitable acid addition salts can be formed by addition of acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, fumaric acid, salicylic acid, citric acid, lactic acid, mandelic acid, tartaric acid, oxalic acid, methanesulfonic acid and other suitable acids known to those skilled in the art.

The present invention also provides pharmaceutical compositions. Suitable compositions comprise a pharmaceutically effective amount of the peptide of the invention or pharmaceutically acceptable salts thereof in admixture with a pharmaceutically acceptable carrier or adjuvant.

The present invention also provides compounds and compositions as defined above for the treatment of pain in mammals, including humans.

The following examples are used to better describe the invention.

Examples

The opioid activity of the peptides was evaluated in vitro using the guinea pig ileum (GPI) longitudinal muscle preparation, and their antinociceptive activity was determined in vivo in PBQ-induced writhing models (peripheral activity) and in the hot plate test (central activity) in rodents. Antagonism of antinociception by the peripheral opioid antagonist N-methylnalorphine and comparison of activities in the writhing and hot plate tests showed that the analgesic effects were predominantly mediated in the periphery. Peripheral analgesia was demonstrated by high potency in the writhing test coupled with low potency in the hot plate test.

PBQ (phenyl-p-benzoquinone)-induced writhing in mice is an assessment of both central and peripheral analgesia. For experimental instructions, see Sigmund et al., Proc. Soc. Exp. Biol. Med., 95, p. 729 (1957). Central analgesia is determined by inhibition of a hot plate response in mice. For experimental instructions, see G. Woolfe and A. MacDonald, J. Pharmacol. Exp.

Ther., 80, p. 300 (1944). Assays measuring opioid receptor binding affinities for µ- and δ-receptors, as well as the GPI and MVD assays, were performed according to the experimental procedure outlined in Schiller et al., Biophys. Res. Commun., 85, p. 1322 (1975).

The compounds of the invention were prepared using solid phase synthesis as set forth below and as is well known to those skilled in the art.

example 1 Solid phase peptide synthesis of opioid peptides

The synthetic peptides were prepared using Rink resin (trade name), 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (Novablochem or Advanced Chemtech) and the corresponding C-terminal Na-Fmoc-L-amino acid residue of each peptide to be synthesized.

All L- and D-amino acids (Novablochem or Advanced Chemtech) had Fmoc-protected α-groups (9-fluorenyl-methyloxycarbonyl) and the following side chain protecting groups: t-butyl ether (tBu) for serine, threonine and tyrosine; t-butyl ester (OtBu) for aspartic acid and glutamic acid; t-butyloxycarbonyl (tBoc) for lysine and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc) for arginine and trityl (trt) for cysteine.

Dimethylformamide (Anachemia) was of dimethylamine-free purity and was treated with activated 4 Å molecular sieves. Piperidine (Advanced Chemtech) was used without further purification. DCC (dicyclohexylcarbodiimide) and HOBt (hydroxybenzotriazole) were purchased from Fluka and Advanced Chemtech, respectively.

Solid phase synthesis was performed manually on Rink resin (trade name). The loading was approximately 0.6 mmol/g. Peptide condensation was performed using: 1) coupling: 2 equivalents each of Fmoc-amino acid, HOBt and DCC in DMF for 1 to 4 hours at room temperature; 2) re-coupling: 1 equivalent each of Fmoc-amino acid, HOBt and DCC; 3) acetylation: 20% (v/v) (CH3CO)2O/DCM for 1 hour at room temperature; 4) N-α-Fmoc deprotection: 20% (v/v) piperidine in DMF for 25 minutes.

Removal of side chain protecting groups (tBu, Boc, Trt, Pmc) and peptide cleavage from the resin were accomplished by a TFA-containing cocktail (v/v) 55/5/40 TFA/anisole/DCM for 90 minutes at room temperature under N2. The peptide was precipitated from diethyl ether, filtered and dried. The crude peptide was purified and analyzed by HPLC on a reverse phase column with gradient elution using 0.06% TFA/H2O and 0.06% TFA/acetonitrile.

Example 2 Hot plate assay Measurement of analgesic activity

For this test, male CD No. 1 mice weighing between 20 and 25 g were used. The mice were weighed, marked and divided into groups of 10.

Mice were usually treated by subcutaneous injection of the compound (or standard or medium) at an injection volume equivalent to 0.1 ml/10 g p.c. (10 ml/kg). If an antagonist such as nalaxone or N-methyl-levallorphan was used, it was administered intraperitoneally 20 minutes before the compound (or standard or medium). The injection volume was also 0.1 ml/10 g p.c. The dose of the antagonist was 10 mg/kg.

Mice were individually assessed for reaction time on the hot plate. The temperature of the hot plate (Sorel, model DS37) was set at 55ºC. Mice were observed for signs of discomfort, such as licking or shaking the paws, escape attempts (jumping off the plate), or trembling. Reaction time was recorded when any of these signs were observed and was recorded in seconds. Each mouse was observed for a maximum period of 30 seconds so that damage to paw tissue was avoided. Mice can be observed at various time intervals after administration of the compound (or medium or standard). Time intervals can be 30, 60, or 120 minutes (or other values).

For each time reading, the mean reaction time of the control group was multiplied by 1.5. The reaction time of each treated mouse was compared to the "control mean x 1.5". If the reaction time was worse than the "control mean x 1.5", the mouse was considered to have no analgesic effect occurred. If the reaction time was superior to the "control mean · 1.5", the mouse was considered to have an analgesic effect. The number of analgesic mice in a group determined the analgesic percentage of the compound for that reading. If the analgesic percentage was worse than 30%, the compound was considered inactive.

Example 3 Curvature assay Measuring the dislocations

The test was carried out on male CD No. 1 mice weighing between 18 and 22 g. The mice were weighed and marked. They were injected intraperitoneally with 0.3 ml/20 g by weight of a solution of phenylquinone at 0.02%. The dislocations that occurred during a period of 15 minutes after the injection were counted. The phenylquinone was injected subcutaneously at 5, 20 or 60 minutes after administration of the compound (or medium or standard). It was injected orally at time intervals of 60 minutes after administration of the compound (or medium or standard).

The 0.02% phenylquinone (2-phenyl-1,4-benzoquinone; Sigma) solution was prepared in the following manner. 20 mg of phenylquinone was dissolved in 5 mL of 90% ethanol (Sigma, Reagent, Alcohol). The dissolved phenylquinone was slowly added to 95 mL of distilled water that was continuously shaken and preheated (not boiled). The phenylquinone solution was protected from light at all times, and a new solution was prepared every day for testing. It is recommended to wait 2 hours before using the phenylquinone solution.

The test can be performed with 5 mice at a time. Each group usually contained 10 mice. If an antagonist such as naloxone was used, it was administered intraperitoneally 20 minutes before the compound (or medium or standard). Table 1

Claims (8)

1. Compound of formula (1): H-Tyr-R2 -Phe-Phe-NH2 (1) with peripheral analgesic effect, wherein R&sub2; D-serine, D-arginine or D-norvaline; or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, wherein R₂ is D-norvaline. 3. A compound according to claim 1, wherein R₂ is D-serine. 4. A compound according to claim 1, wherein R₂ is D-arginine. 5. A compound according to claims 1 to 4 for use as a medicament. 6. Pharmaceutical composition with peripheral analgesic activity, comprising an effective amount of at least one compound according to claims 1 to 4 in admixture with a pharmaceutically acceptable carrier. 7. Use of the compound according to claims 1 to 4 for the preparation of a peripheral analgesic for the treatment of pain. 8. Use of H-Tyr-D-Ala-Phe-Phe-NH₂ for the manufacture of a peripheral analgesic for the treatment of pain.